Our recent findings made during this funding period have revealed an unexpected dependence of canonical Burkitt's Lymphomas (BLs) and Wp-restricted BLs on EBV's BART miRNAs (Vereide and Sugden, 2011;Vereide et al., submitted). We have also found that EBV's miRNAs promote transformation of newly infected B-cells (Seto et al., 2010;Vereide et al., submitted). We propose to identify the mRNAs that are regulated by EBV's miRNAs to drive tumor maintenance and promote transformation. EBV encodes 25 pre-miRNAs which can yield 50 mature miRNAs. These miRNAs have predicted seed sites in more than 30% of all human mRNAs. However, many studies, including our own, indicate that the vast majority of these mRNAs are not regulated by EBV's miRNAs (Kuzembayeva et al. submitted;Dolken et al. 2010). It is therefore essential to develop functional assays to allow characterization of presumptive targets of miRNAs in order to learn if the subtle differences in their expression mediated by miRNAs have functional consequences. We have developed functional assays for tumor maintenance and transformation dependent on EBV's miRNAs when the miRNAs are expressed at physiological levels. This latter qualification is important because miRNAs act in a dose-dependent manner, and EBV's miRNAs are often expressed at low levels (Pratt et al., 2009). We propose to identify minimal subsets of EBV's miRNAs that regulate a given phenotype and then develop matched pairs of cells that do and do not express these miRNAs from EBV plasmids. The mRNAs in the RISCs immunoprecipitated from these pairs of cells will be identified and enumerated by deep sequencing, sorted bioinformatically, and their 3'UTRs tested in reporter assays for regulation by specific viral miRNAs. We have used all of these assays successfully in our identification of caspase 3 as a target for BART miRNAs in canonical BLs (Vereide et al. submitted). The mRNAs found by this set of assays will then be tested functionally for potential contributions to maintaining lymphomas and transforming primary B-cells.

Public Health Relevance

Epstein - Barr virus (EBV) causes lymphomas and carcinomas in people throughout the world. No vaccine is available for preventing infection by EBV. We are identifying and characterizing those EBV genes that allow tumors to survive and proliferate to elucidate viral targets for developing anti-viral, anti-tumor therapies. These studis are also revealing how EBV, which is a paradigm for human tumor viruses, sustains tumors. EBV is exceptional for the number of miRNAs it encodes. We have now found that these small RNAs contribute both to tumor maintenance and to transformation of newly infected B-cells and propose to elucidate their mechanisms of driving this tumorigenesis and transformation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA070723-16A1
Application #
8503777
Study Section
Virology - A Study Section (VIRA)
Program Officer
Daschner, Phillip J
Project Start
1996-08-15
Project End
2018-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
16
Fiscal Year
2013
Total Cost
$304,213
Indirect Cost
$78,255
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Chiu, Ya-Fang; Sugden, Bill (2016) Epstein-Barr Virus: The Path from Latent to Productive Infection. Annu Rev Virol 3:359-372
Albanese, Manuel; Tagawa, Takanobu; Bouvet, Mickaël et al. (2016) Epstein-Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells. Proc Natl Acad Sci U S A 113:E6467-E6475
Tagawa, Takanobu; Albanese, Manuel; Bouvet, Mickaël et al. (2016) Epstein-Barr viral miRNAs inhibit antiviral CD4+ T cell responses targeting IL-12 and peptide processing. J Exp Med 213:2065-80
Steinbrück, Lisa; Gustems, Montse; Medele, Stephanie et al. (2015) K1 and K15 of Kaposi's Sarcoma-Associated Herpesvirus Are Partial Functional Homologues of Latent Membrane Protein 2A of Epstein-Barr Virus. J Virol 89:7248-61
Hammerschmidt, Wolfgang (2015) The Epigenetic Life Cycle of Epstein-Barr Virus. Curr Top Microbiol Immunol 390:103-17
Chakravorty, Adityarup; Sugden, Bill (2015) The AT-hook DNA binding ability of the Epstein Barr virus EBNA1 protein is necessary for the maintenance of viral genomes in latently infected cells. Virology 484:251-8
Kuzembayeva, Malika; Hayes, Mitchell; Sugden, Bill (2014) Multiple functions are mediated by the miRNAs of Epstein-Barr virus. Curr Opin Virol 7:61-5
Vereide, D T; Seto, E; Chiu, Y-F et al. (2014) Epstein-Barr virus maintains lymphomas via its miRNAs. Oncogene 33:1258-64
Sugden, Bill (2014) Epstein-Barr virus: the path from association to causality for a ubiquitous human pathogen. PLoS Biol 12:e1001939
Shrestha, Prabha; Sugden, Bill (2014) Identification of properties of the Kaposi's sarcoma-associated herpesvirus latent origin of replication that are essential for the efficient establishment and maintenance of intact plasmids. J Virol 88:8490-503

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